1. Field of the Invention
This invention relates to the isolation and characterization of novel Classical Swine Fever Virus (CSFV) virulence determinants within the E2 structural glycoprotein and utilization of these novel virulence determinants to design live attenuated CSF vaccines.
2. Description of the Relevant Art
Classical swine fever (CSF) is a highly contagious disease of swine that can be either acute or chronic in nature (van Oirschot, J. T. 1986. In: Diseases of Swine, 6th edition, Leman et al., eds., Iowa State University Press, Ames, Iowa, page 289). The etiological agent, CSF virus (CSFV), is a small, enveloped virus with a positive, single-stranded RNA genome and, along with bovine viral diarrhea virus (BVDV) and border disease virus (BDV), is classified as a member of the genus Pestivirus within the family Flaviridae (Hulst et al. 2001. J. Virol. 75: 9585-9595). The 12.5 kb CSFV genome contains a single open reading frame which encodes a 4000 amino acid polyprotein and ultimately yields 11 to 12 final cleavage products (NH2-Npro-C-Erns-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH) through co- and posttranslational processing of the polyprotein by cellular and viral proteases (Rice, C. M. 1996. In: Fundamental Virology, 3rd edition, Fields and Howley, eds., Lippincott Raven, Philadelphia, pp. 931-959).
Virulence and host range phenotypes vary among CSFV isolates and between pestiviruses. Infection with highly virulent CSFV strains leads to death in infected animals, whereas isolates of moderate to low virulence induce a prolonged chronic disease (van Oirschot, supra). In addition, BVDV and BDV, while etiologic agents of diseases in bovine and ovine species, respectively, can also infect swine without inducing clinical disease (van Oirschot, supra). Despite availability of genomic sequences from CSFV of differing virulence phenotypes, BVDV, and BDV, the genetic basis of CSFV virulence in the natural host remains poorly understood (van Oirschot, supra). The use of reverse genetics has enabled the identification of viral determinants of virulence facilitating development of candidate live attenuated CSF vaccines (Mayer et al. 2004. Vaccine 22: 317-328; Meyers et al. 1999. J. Virol. 73: 10224-10235; Moorman et al. 1996. J. Virol. 70: 763-770; Moser et al. 2001. Virus Genes 23: 63-68; Risatti et al. 2005a. J. Virol. 79: 3787-3796; Risatti et al. 2005b. Virology 343: 116-117; Ruggli et al. 1996. J. Virol. 70: 3478-3487; Tratschin et al. 1998. J. Virol. 72: 7681-7684; van Gennip et al. 2000. Vaccine 19: 447-459).
The capsid protein, and glycoproteins Erns, E1, and E2 are the structural components of the CSFV virion with E1 and E2 anchored to the envelope by their carboxyl termini and Erns loosely associated with the viral envelope (Thiel et al. 1991. J. Virol. 65: 4705-4712; Weiland et al. 1990. J. Virol. 64: 3563-3569; Weiland et al. 1999. J. Gen. Virol. 80: 1157-1165). All three glycoproteins have been associated with CSFV virulence (Meyers, supra; Risatti et al. 2005 a, b, supra).
E2 glycoprotein is considered essential for CSFV replication, as virus mutants containing partial or complete deletions of the E2 gene have proven non-viable (van Gennip et al. 2002. Vaccine 20: 1544-1556). E2 has been implicated, along with Erns (Mayer et al., supra) and E1 (Wang et al. 2004. Virology 330: 332-341), in viral adsorption to host cells; indeed, chimeric pestiviruses exhibit infectivity and cell tropism phenotypes consistent with those of the E2 gene donor (van Gennip et al. 2000, 2002, supra). E2 is the most immunogenic of the CSFV glycoproteins (Konig et al. 1995. J. Virol. 69: 6479-6486; Weiland et al. 1990, supra), inducing neutralizing antibodies and protection against lethal challenge. CSFV E2 also contains, between residues 829 and 837, an epitope recognized by monoclonal antibody (mAb) WH303 (Lin et al. 2000. J. Virol. 74:11619-11625), a reagent which fails to react with BVDV or BDV E2 and is routinely used for CSF diagnostics.
Here we report the effects of mutations within the WH303 epitope of CSFV E2, mutations which change the amino acid sequence of the virulent Brescia CSFV progressively toward the homologous amino acid sequence of BVDV strain NADL, demonstrating an additive effect for viral virulence in swine and complete attenuation after six amino acid changes. Such attenuate viruses permit the rational design of live attenuated CSF vaccines. Animals infected with virus mutants were protected when challenged with virulent Brescia virus at 3 and 21 days post vaccination. Modification at this site within the WH303 epitope allows development of a diagnostic test to differentiate vaccinated from infected animals.